DS0404 - Innovation biomédicale

Haematopoietic specification in the human embryo: toward de novo generation of Hematopoietic Stem Cells – ACEBLOOD

Understanding the in vivo blood formation: towards an in vitro therapeutic production

The aim of ACEBLOOD proposal is to improve the understanding of cellular and molecular events leading to the specification and activation of a hematopoietic program during embryonic development.

Genetic programs at the base of hematopoietic stem cell generation

The continuous generation of blood cells throughout life relies on the existence of hematopoietic stem cells generated during embryogenesis. These cells are multipotent, capable of long-term self-renewal and can be transplanted to patients with inherited or acquired blood diseases. However, the availability of immuno-compatible donors remains a major problem. <br />Despite progress made over the last decade in the search of alternative sources to overcome this shortage, the reprogramming of pluripotent cells now only results in the in vitro generation of progenitors whose abilities of self-renewal and multipotency remain, however, limited.<br />The aim of ACEBLOOD proposal is to improve the understanding of cellular and molecular events leading to the specification and activation of a hematopoietic program during embryonic development. Indeed, a clearer understanding of the embryonic signaling and morphogenetic processes might suggest additional molecular factors that could make the «reprogramming process« more robust and efficient and therefore open on the possibility of generating and amplifying HSCs in vitro. <br />Based on these considerations, our project is perfectly adequate for the societal challenge of developing a disease- and patient-specific approach to gene and cell therapy a major goal of regenerative medicine.

Partner 1 has shown in vitro the aptitude of transduced HSC by the lentiviral vector to proliferate and differentiate in different blood cell population demonstrating the conservation of their multipotence. Furthermore, she has carried out the enzymatic and dissection methods necessary to obtain isolated cells from embryonic tissues and in association with the Microarray & Sequencing and Bioinformatics Platforms has defined the strategy to perform bioinformatics analysis of rare cells isolated from human embryos.
Partner 2 has improved the conditions of human cell transplantation in the zebrafish embryo. Different culture media have been compared in order to find the best compromise allowing human cell and zebrafish embryos survival, reducing the incidence of death of the embryos by adding a cocktail of antibiotics and defining the optimal temperature. Finally, we optimized a live imaging under a dissecting scope during one week in order to track transplanted cells fate without compromising embryo development and survival.
Partner 3 has set up the conditions allowing a rapid transduction of HSC for their fluorescent labelling by lentiviral vectors. The issue of the rapidity of the transduction time is crucial to avoid extended culturing of HSC which might lead to artefactual differentiation in vitro. Also, the vectors have been engineered in such a way as to express a nuclear variant of the GFP under the control of a strong promoter (CMV in the final version, EF1-? in the initial one). The conditions allowing efficient labelling of the cells for the lowest MOI (multiplicity of infection -actually transduction by the lentiviral vectors-) have been established. This is a crucial point since low MOI will lead to inefficient labelling while high MOI, leading to integration of multiple copies of the retroviral genome, can lead to unwanted insertional mutagenesis of many genes, possibly modifying the behavior of the transduced HSC.

(a) First, to visualize transplanted cells in zebrafish embryos, we have developed a rapid method of lentiviral transduction. We have then engrafted transduced cells into zebrafish embryos and deciphered the reason of engraftment failure. We thus established conditions for reproducible hematopoietic engraftments. Our results have demonstrated for the first time that the hematopoietic engraftment success depends on the depletion of the zebrafish primitive macrophages.
(b) Second, based on a transcriptomic analysis we have identified that the gene COTL1 – known to be involved in the synthesis of leukotrienes – is expressed in the intra-aortic HSCs, as well as in the hematopoietic progenitors of the yolk sac during human development. This result opens the way to further explore the role of leukotrienes in the hematopoietic specification.

There is no doubt that these results will be valued through
- the redaction of 3 scientific articles. The first one will be submitted soon
- the presentation at scientific congresses
A very important work describing how to isolate human HSCs from human embryos has been established.
The stable labelling of human HSCs by a lentivirus as well as the demonstration of the conservation of their totipotency is also established.
We have demonstrated the interest of using an alternative model, the zebrafish embryo, an ethically accepted model by regulatory authorities and which replaces the use of mammalian models such as mice and rats. Its transparency allows real-time monitoring of the behaviour and fate of stem cells. This work is ongoing and will allow the study of other stem cells for therapeutic purposes.
Finally, a large number of genes have been identified and opens new perspectives for the study of the emergence and fate of human HSCs in vitro.
Of particular interest is the discovery that COTL, a gene involved in the production of the leukotrienes , is expressed in all hemogenic sites during embryonic development, such as the AGM and the yolk sac.
Although preliminary, these results are very encouraging and prompt towards a more in-depth analysis aimed at developing of a pharmacological approach of loss- and gain-of-function to investigate the role of leukotriene synthesis in the hematopoietic emergence.

Part of these results has been presented as posters at several national and international scientific meetings.
These works are the subject of three scientific articles which are in the process of being finalized for publication.

The continuous generation of blood cells throughout life relies on the existence of haematopoietic stem cells (HSC) generated during embryogenesis. Clinically, these cells are the relevant component of bone marrow transplants, patients with inherited and acquired blood diseases. However, the availability of immune-compatible donors remains a problem. Transplant engraftment failure, graft-versus-host disease (GVHD) and delayed reconstitution still remain significant causes of morbidity and mortality following bone marrow transplantation.
Recently, the possibility to activate primitive pluripotent genes within adult human cells that take them back in time to pluripotent state (called induced pluripotent stem cells, iPS) provided a major breakthrough for the field of regenerative medicine. The in vitro production of HSCs from iPS cells could provide a source of transplantable cells for the treatment of sickle cell anaemia, thalassaemia, leukaemia and other diseases also raising the possibility of autologous transplantation. Although the generation of specific types of haematopoietic cells for specific applications is now becoming feasible, generating robust numbers of bona fide HSCs that are capable of long-term self-renewal and with normal lineage potential remains impossible so far. This suggests that key specification requirements are still unknown.
Understanding how HSC specification occurs natively during embryonic development and trying to carefully reproduce these events is one means of trying to develop improved techniques for directed differentiation protocols. Adult-type HSC are first generated in the aorta-gonad-mesonephros (AGM) region between days 27 and 40 of human embryonic development, but an elusive blood forming potential is present earlier in the underlying splanchnopleura (Sp). The origin of blood cells has been the subject of an intense scientific debate during the last decade. Although it is relatively well accepted that the first HSCs are generated in the AGM through a haemogenic endothelium, a process evolutionary conserved, the direct precursor of this cell type in the embryo, either a mesenchyme or a haemangioblast, remains to be clearly identified.
Here, we examine the cellular origin of HSC in the human embryo. The study is focused on the angiotensin-converting enzyme (ACE) that we have shown to identify pre-haematopoietic cells inside the early human embryo. The potential ability and fate of ACE+ cells will be analysed by in vitro approaches. The fate and behaviour of ACE+ cells will also be analysed in vivo by transplant into the zebrafish embryo (Danio rerio) that, being transparent, allows the direct visualization of haematopoietic cells into live animals, especially using fluorescent transgenic strains marking the tissues of interest. Sorted ACE+ cells will be engineered by lentiviral transduction (following a procedure lasting only 4 hours, a crucial parameter when one manipulates stem cell progenitors) to constitutively express fluorescent protein markers, and will then be transplanted in the zebrafish embryo at early stages in blastula or, later, directly in the blood circulation.
The transcriptome profile of pre-haematopoietic ACE+ cells sorted from human embryo will also be characterised, in collaboration with the Microarray and Sequencing Platform at the IGBMC and carried out on single cells using the Fluidigm platform (C1™ Single-Cell Auto Prep System and BioMark™ HD System).
The ultimate goal of this study is to obtain a comprehensive panel of human embryonic specific genes that may be responsible for activating a haematopoietic program.

Project coordination

Manuela Tavian (UMR-S949 INSERM Université de Strasbourg)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


UM2 UMR5235 - DIMNP Dynamique des interactions membranaires normales et patholgiques
INSERM UMR-S949 UMR-S949 INSERM Université de Strasbourg
CNRS UPR 9002 Retroviruses and Molecular Evolution CNRS-UPR 9002 Institut de Biologie Moléculaire et Cellulaire

Help of the ANR 404,220 euros
Beginning and duration of the scientific project: October 2014 - 36 Months

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